Solubilising polysaccharides substituted with dydrophilic and hydrophobic groups

ABSTRACT

This invention relates to novel carbohydrate polymers with hydrophobic and hydrophilic side-groups suitable for solubilising, for example, hydrophobic drugs. The chain length of the carbohydrate polymeric backbone, and the type and number of the hydrophobic and hydrophilic side-groups are specifically chosen to improve the solubility properties of the carbohydrate polymers.

FIELD OF THE INVENTION

This invention relates to novel carbohydrate polymers with hydrophobicand hydrophilic side-groups suitable for solubilising, for example,hydrophobic drugs. The chain length of the carbohydrate polymericbackbone, and the type and number of the hydrophobic and hydrophilicside-groups are specifically chosen to improve the solubility propertiesof the carbohydrate polymers.

BACKGROUND OF THE INVENTION

Soluble polymers bearing pendant amphiphilic or hydrophobic groups,commonly known as polysoaps, have been studied for a number of years andnumerous applications proposed¹ based on exploiting their solubilisationcapacity for hydrophobic molecules^(2, 3). These compounds formintramolecular micelles^(4, 5), usually several per molecule⁶, and theirsolubilisation capacity is not lost on dilution¹ unlike small molecularweight micelles⁷, making them especially useful as solubilisers.Although these molecules are well known they have not been exploited toany great extent as pharmaceutical solubilisers.

Hydrophobic drugs are those drugs, which are practically insoluble inwater. The definition of “practically insoluble” used by the BritishPharmacopoeia is used here and is defined as a situation where 1 g ofsuch material requires more than 10,000 millilitres of solvent (e.g.water) to be solubilised⁸ or alternatively a material which has asolubility of less than 0.1 mg mL⁻¹ in water.

A few pharmaceutical solubilisers have been reported usinghydrophobically and hydrophilically modified chitosans, namely: N-acyl6-sulphated chitosans¹⁰, quaternary ammonium palmitoyl glycol chitosan⁸and alkylated poly(L-lysine citramide)¹¹, although the effects ofdepolymerisation of the carbohydrate backbone was not explored in thework done on these carbohydrates^(8, 9, 11). However, two differentmolecular weights of N-lauroyl 6-carboxymethyl chitosan, with onemolecular weight class being investigated at two different levels oflauroyl and carboxymethyl substitution, have been reported by Miwa andothers¹¹ as “micellar” carriers of the hydrophobic drug paclitaxel. Thepaclitaxel formulation described by these workers however is prepared bythe probe sonication of N-lauroyl 6-carboxymethyl chitosan andpaclitaxel in a 10% v/v ethanol solution. The removal of ethanol bydialysis was attempted but not confirmed by these workers and finalN-lauroyl 6-carboxymethyl chitosan—paclitaxel formulations weredescribed as “turbid” with particle size ranges of between 30 and 300 nmand a mean particle size of between 32 and 82 nm. In contrast, theinvention disclosed herein relates to the attributes of a solubilisingpolymer which produces optically clear solutions (devoid of appreciablelight scattering) when hydrophobic drugs are added to an aqueous phase(devoid of organic solvents) in the presence of the solubilisingpolymer. Miwa and others on the other hand report that the precursor toN-lauroyl 6-carboxymethyl chitosan which contains no hydrophobicchain—carboxymethyl chitin “yielded a clear solution and the scatteringphenomena detected in the case of micellar solution were not observed inthe carboxymethyl chitin solution”¹¹. The present invention thus differsfrom that reported by Miwa and others¹¹ in that an aqueous opticallyclear solution is prepared from the polymer and appropriateconcentrations of poorly soluble drugs. Organic solvents are also notrequired in the preparation of the present solutions.

Hydrophobically modified chitosans soluble in dilute acid solutions havealso been reported^(12, 13). Other hydrophobically modifiedcarbohydrates have been reported to yield particulate¹⁴⁻¹⁹ dispersionsin aqueous media as opposed to water soluble materials¹³—namelypalmitoyl glycol chitosan^(14, 15), deoxycholic acid modifiedchitosan^(16, 17) and cholesterol bearing pullulans^(18, 19) oralternatively aqueous insoluble gel-like materials^(20, 21).

While the advantageous influence of depolymerisation, controlledhydrophilic substitution, and controlled hydrophobic substitution ofcarbohydrates on the production of an optically clear solution withhydrophobic drugs has not previously been described, reports on theindividual influences of depolymerisation, hydrophobic substitution andhydrophilic substitution on polymer behaviour can be found in theliterature. There is an indirect relationship between the length ofhydrophobic pendant groups and water solubility in the case ofhydrophobised starches²² and hydrophobised ethyl celluloses²⁰. Thisparameter also has a direct influence on the degree of polymeraggregation when in solution in the case of amphiphilic chitosans⁹ anddextrans²³ as well as on the solubilising properties of hydrophobicallymodified chitosans⁹. The degree of hydrophobic substitution has alsobeen reported to have an indirect influence on the aqueous solubility ofstarch derivatives²² and affects the flow properties of hydroxypropylguar gums.

The balance of hydrophobic and hydrophilic substitution has also beenreported to affect a number of polymer properties. An increase inhydrophobic substitution from 20 substituents in every 100 monomers to90 substituents in every 100 monomers with an associated decrease in thelevel of carboxymethyl substituents from 200 substituents in every 100monomers to 140 substituents in every 100 monomers decreased theassociation of paclitaxel with the N-lauroyl 6-carboxymethyl chitosancolloids¹¹, indicating that a more hydrophobic polymer promotedassociation of paclitaxel with the chitosan based colloid. The balancebetween the level of hydrophobic and hydrophilic modification alsoaffected the flow properties of amphiphilic hydroxyethylcelluloses²⁴ andan optimum hydrophobic modification level for amphiphilic chitosans hasbeen identified when these materials are used to prevent wool shrinkageduring washing²⁵.

With regard to amphiphilic polymer molecular weight alone this has beenshown to have an indirect effect on the emulsifying activity ofhydrophobised starches²⁶ and an optimum molecular weight has beenidentified for amphiphilic chitosans bearing deoxycholic acid pendantgroups in the context of DNA—chitosan nanoparticles fabricated for genedelivery²⁷. Also the molecular weight of hydrophobised (C6-acyl)dextrans influenced the phase separation of these systems with the highmolecular weight material being more likely to phase separate²³. Themolecular weight of hydrophobic hydroxypropyl guar gums²⁸ was found toinfluence their flow properties. However, the molecular weight ofN-lauroyl 6-carboxymethyl chitosan polymers did not affect their abilityto encapsulate paclitaxel within the chitosan based colloid¹¹.

Returning to the work of Zhang and others on acyl dextrans²³, the mainpurpose of this work was to “prepare a family of polymer pairs in whichthe compatibility in aqueous solution could be varied in subtle ways”with a view to applying the results to the development of adhesives. Theauthors conclude that the tendency for dextran and hydrophobicallymodified dextran to phase separate increases with molecular weight,degree of hydrophobic substitution and hydrophobic chain length²³. Itshould be noted that Zhang and others²³ did not explore the effect ofmolecular weight on the phase separation of hydrophobically andhydrophilically modified dextrans.

It is an object of embodiments of the present invention to obviate ormitigate at least one or more of the aforementioned problems.

It is a further object of embodiments of the present invention toprovide a polymer for solubilising hydrophobic materials such as drugs.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided asolubilising carbohydrate polymer of average molecular weight of about2-30 kD according to the following formula:

wherein m is 0.01% to 10.00%;

-   -   n is 0.01% to 99.98%;    -   p is 0.00% to 99.98%;

X is any linear or branched, substituted or unsubstituted, or cyclo formof an alkyl, alkenyl, alkynyl, aryl, amine, amide, alcohol or acylgroup;

R′, R″, R′″ are independently any linear or branched, substituted orunsubstituted, or cyclo form of an alkyl, alkenyl, alkynyl, aryl, amine,amide, alcohol, acyl group, any sugar substitutent or oligo polyoxaC₁-C₃ alkylene units; and

R₁, R₂ and R₃ are independently any linear or branched, substituted orunsubstituted, or cyclo forms of any alkyl, alkenyl, alkynyl, aryl oracyl group.

It is understood that m+n+p will be equal to 100%. It should also beunderstood that m, n and p may form any arrangement in the solubilisingcarbohydrate polymer. The arrangement of the m, n and p units maytherefore be random or in a block copolymer form such as mnpmnpmnp etc.This is identified in the structure shown above by the dashed linebetween the different monomer units.

The carbohydrate polymer may be positively charged with a counter ion.The counter ion may be represented by any negative ion. Typically thecounter ion may be ions of any of the following: chloride, iodide,acetate and glucoronide.

If the monomer unit identified by n is uncharged (i.e. the hydrophilicside group is uncharged) then the carbohydrate polymer may be unchargedand there will therefore be no counter ion.

Typically, X may be selected from any of the following linear orbranched, substituted or unsubstituted, or cyclo groups: C₁-C₃₀; C₈-C₂₄;or C₁₂-C₁₈.

The X group may be selected from any of the following: any type of fattyacid derivative of, for example, stearic acid, oleic acid, palmiticacid; N-hydroxysuccinimide acid and any other activated acyl compounds;and anhydrides.

In particular, X may be CH₃(CH₂)₁₄CONH, or CH₃(CH₂)₁₅NH.

R₁, R₂ and R₃ may independently be any linear or branched, substitutedor unsubstituted, or cyclo form of the following alkyl, alkenyl,alkynyl, aryl or acyl groups: C₁-C₃₀; C₁-C₁₂; C₁-C₆; or C₁.

Typically, R₁, R₂ and R₃ may be C₁-C₄ linear alkyl groups.

Conveniently, all of R₁, R₂ and R₃ may be CH₃.

On some monomers the C2 nitrogen may not be fully substituted and may bepresent as a secondary or tertiary amine.

R′, R″ and R′″ may independently be any linear or branched, substitutedor unsubstituted, or cyclo form of the following alkyl, alkenyl,alkynyl, aryl, amine, amide, alcohol or acyl groups: C₁-C₃₀; C₁-C₁₂; andC₁-C₆.

Typically, R′, R″ and R′″ may be C₁-C₄ linear glycol based groups.

Typically, R′, R″ and R′″ are any of the following sugar substituents:glucose, galactose, fructose and muramic acid.

R′, R″ and R′″ may be oligo polyoxa C₁-C₃ alkylene units such asethylene glycol oligomers.

All of R′, R″ and R′″ may be CH₂OCH₂CH₂OH or CH₂CH₂OH.

The carbohydrate polymer starting material to which additionalhydrophobic and hydrophilic groups are attached may have an averagemolecular weight of about 2 to 30 kD. Preferably, the carbohydratepolymer starting material has a molecular weight of about 5 to 17 kD.

Conveniently, the X group may be hydrophobic.

Conveniently, the R₁, R₂ and R₃ groups form a quaternary ammonium groupwhich is hydrophilic.

Hydrophilic groups are groups, which are well hydrated by water andassociate on a molecular level with water. A further non-ionichydrophilic group may replace NR₁R₂R₃, providing R′, R″ and R′″ areequal to CH₂0-Y and Y is a hydrophilic substituent. In that case bothhydrophilic substituents on the carbohydrate polymer may be selectedfrom mono and oligo hydroxy C1-C6 alkyl, mono and oligo hydroxysubstituted C2-C6 acyl, C1-C2 alkoxy alkyl optionally having one or moreof the hydroxy groups substituted on the alkoxy or alkylene groups,oligo or poly-(oxa C1-C2 alkylene), preferably polyethylene glycolcomprising up o 120 ethylene oxide units (i.e. a molecular weight of 5,000), and C1-C4 alkyl (oligo or poly oxa C1-C3 alkylene) optionallyhydroxy substituted preferably oligo or polyglycerol ethers; wherein thereplacement group for NR₁R₂R₃ is joined via an ether linkage to asaccharide unit of the polysaccharide. The acyl group may contain alkyl,alkenyl or alkynyl groups.

The R′, R″ and R′″ groups may also be hydrophilic.

Typically, the ratio of m:n:p may have the following range: 0.1:1:98.9to 9:91:0; 1:5:96 to 8:50:42; or 3:10:87 to 5:19:76.

The total number of monomer units of m+n+p may be about 10 to 100.Preferably, the total number of monomer units of m+n+p may be less thanabout 200.

Typically, the number of X groups may not exceed 10 for every 100monomer groups in the carbohydrate backbone.

The solubilising carbohydrate polymer may also contain additionaltargeting groups such as peptides, antibodies and other ligands, forexample, folate and transferrin ligands which may allow the polymer totarget endogenous receptors and thus target its drug payload to suchendogenous receptors at the site of pathology.

According to a second aspect of the present invention there is provideda method of forming a solubilising carbohydrate polymer according to thefirst aspect wherein the method comprises;

depolymerising a carbohydrate polymer to form depolymerisedcarbohydrate;

reacting the depolymerised carbohydrate with a first reactive compoundto form hydrophobic side-groups on the carbohydrate backbone and thusform hydrophobically substituted depolymerised carbohydrate; and

adding a second reactive compound to the hydrophobically substituteddepolymerised carbohydrate to quaternarise an amine group and therebyform the solubilising carbohydrate polymer.

The carbohydrate polymer may be selected from the following: glycolchitosans, dextrans, alginic acids, starches, dextran, guar gums and allother carbohydrate polymers.

The carbohydrate polymer may be depolymerised with any of the following:an acid, a base, or enzyme.

The acid used to depolymerise the carbohydrate polymer may be selectedfrom any of the following: HCl, H₂SO₄, HNO₃ or HF.

The carbohydrate polymer may be depolymerised for a few days, forexample, 48 hours, then isolated and subjected to furtherdepolymerisation dependent on the average molecular weight ofsolubilising carbohydrate polymer required.

The average molecular weight of carbohydrate polymer to be depolymerisedis about 3 to 30 kD and is preferably about 15 kD.

The first reactive compound which forms the hydrophobic side-groups onthe depolymerised carbohydrate polymer may be selected from any of thefollowing: any type of fatty acid derivative of, for example, stearicacid, oleic acid, palmitic acid; organo halides such as alkyl, alkenyl,alkynyl, cyclic or non-aromatic halides, acyl chlorides, anhydrides,N-hydroxysuccinimide and other activated acyl compounds capable of beingattacked on the C1 carbon by a compound capable of nucleophilic attack.By nucleophilic attack is meant compounds which attack atoms with a lowelectron density. The acyl groups may also contain an alkyl, alkenyl oralkynyl group.

Preferably, the first reactive compound which forms the hydrophobicside-groups on the depolymerised glycol chitosan may be selected formany of the following: hexadecyl bromide, dodecyl bromide, myristic acidN-hydroxysuccinimide.

Preferably, the fatty acid derivative may be palmitic acidN-hydroxysuccinimide; palmitic acid benzotriazole carbonate;palmitaldehyde; palmitoyl chloride; and palmitic acid p-nitro phenylcarbonate.

The second reactive compound may be an organo halide wherein the organomay be selected from any linear or branch, substituted or unsubstituted,or cyclo form of any alkyl, alkenyl, alkynyl, aryl, amine, amide,alcohol or acyl group.

Typically, the second reactive compound may be any linear or branched,substituted or unsubstituted, or cyclo form of the following alkyl,alkenyl, alkynyl, aryl, amine, amide, alcohol or acyl groups: C₁-C₃₀;C₁-C₁₂; C₁-C₆; or C₁.

Typically, the organo group of the organo halides may be short chainlinear alkyl groups.

The organo group of the organo halides may be CH₃.

The solubilising carbohydrate polymer obtained may be purified by columnchromatography, dialysis and freeze drying.

According to a third aspect of the present invention there is provided acarbohydrate polymer according to the first aspect and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M, or preferably 0.05 Mphosphate buffer or 0.9% saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethyleneglycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, anti-microbial, anti-oxidants,chelating agents, inert gases and the like.

Typically, the ratio of carbohydrate polymer to pharmaceuticalacceptable carrier ranges from 0.05 wt. % to 10 wt. %.

According to a fourth aspect of the present invention there is provideda pharmaceutical composition comprising a carbohydrate polymer accordingto a first aspect and a drug.

The drug may be poorly soluble in aqueous solvents such as water.

The drug may be selected from any of the following: prednisolone;cyclosporine; oestradiol, testosterone, drugs with multicyclic ringstructures which lack polar groups such as paclitaxel and drugs such asetoposide.

Typically, the ratio of carbohydrate polymer to the drug may be 10 wt. %: 5000 wt. %.

Typically, the ratio of carbohydrate polymer to drug to pharmaceuticallyacceptable carrier may be about 1 mg:1-5 mg:1 g.

The pharmaceutical composition may be in the form of any of thefollowing: tablets, suppositories, liquid capsule, powder form, or aform suitable for pulmonary delivery.

When tablets are used for oral administration, typically used carriersinclude sucrose, lactose, mannitol, maltitol, dextran, corn starch,typical lubricants such as magnesium stearate, preservatives such asparaben, sorbin, anti-oxidants such as ascorbic acid, α-tocopheral,cysteine, disintegrators or binders. When administered orally ascapsules, effective diluents include lactose and dry corn starch. Aliquid for oral use includes syrup, suspension, solution and emulsion,which may contain a typical inert diluent used in this field, such aswater, in addition, sweeteners or flavours may be contained.

Suppositories may be prepared by admixing the compounds of the presentinvention with a suitable non-irritative excipient such as those thatare solid at normal temperature but become liquid at the temperature inthe intestine and melt in the rectum to release the active ingredient,such as cocoa butter and polyethyleneglycols.

The dose can be determined on age, body weight, administration time,administration method, combination of drugs, the level or condition ofwhich a patient is undergoing therapy and other factors. While the dailydoses may vary depending on the conditions and body weight of patients,the species or active ingredient, and administration route, in the caseof oral use, the daily doses may be about 0.1-2 mg/person/day,preferably 0.5-100 mg/person/day.

According to a fifth aspect of the present invention there is provided amethod of dissolving poorly soluble drugs in a carbohydrate polymerwherein the solubilising carbohydrate polymer has a specificallydesigned average molecular weight, and specific type and amount ofhydrophilic and hydrophobic side-groups substituted on a carbohydratepolymeric backbone whereby on dissolving a poorly soluble drug in thesolubilising carbohydrate polymer a substantially clear solution isobtained.

By substantially optically clear solution herein is meant an opticallyclear solution which is devoid of appreciable light scattering and isclear to the naked eye.

By poorly soluble drugs is meant where one gram of a drug requires morethan 10,000 ml of solvent (water) to be solublised. Alternatively, thismeans a drug which has a solubility of less than 0.1 mg mL⁻¹ in water.

Typically, the solubilising carbohydrate polymer is selected from anyderivatives of the following: chitosans, dextrans, alginic acids,starches, dextran, guar gums and all other carbohydrate polymers.

In particular, the carbohydrate polymer according to the first aspectmay be used.

The poorly soluble drug may be selected from any of the following:cyclosporin, steroids such as prednisolone, oestradiol, testosterone,drugs with multicyclic ring structures which lack polar groups such aspaclitaxel and drugs such as etoposide.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the accompanying drawings in which:

FIG. 1 a is a representation of quaternary ammonium palmitoyl glycolchitosan (GCPQ);

FIG. 1 b is a representation of quaternary ammonium hexadecyl glycolchitosan (GCHQ);

FIG. 2 a is a representation of the haemolytic activity of GCPQ preparedfrom glycol chitosan which has been subjected to an initial 48 hoursacid degradation and a further 8 hours acid degradation, followed byacylation with palmitic acid N-hydroxysuccinimide and alkylation withmethyl iodide (i.e. GCPQ488);

FIG. 2 b is a representation of the haemolytic activity of GCPQ488 andcyclosporine in a 1:1 ratio;

FIG. 2 c is a representation of the haemolytic activity of GCHQ preparedfrom glycol chitosan which has been subjected to an initial 48 hoursacid degradation and a further 8 hours degradation, followed byalkylation with hexadecyl bromide and alkylation with methyl iodide(i.e. GCHQ488);

FIG. 2 d is a representation of the haemolytic activity of GCHQ488 andprednisolone in a 1:5 ratio;

FIG. 3 a is a representation of the cyctotoxicity to A549 cell linesafter incubation with GCPQ488;

FIG. 3 b is a representation of the cyctotoxicity to A549 cell linesafter incubation with GCPQ488 and cyclosporine;

FIG. 3 c is a representation of the cyctotoxicity to A549 cell linesafter incubation with GCHQ488;

FIG. 3 d is a representation of the cytotoxicity to A549 cell linesafter incubation with GCHQ488 and prednisolone in a 1:5 ratio;

FIG. 3 e is a representation of the cyctotoxicity to A431 cell linesafter incubation with GCPQ488 and cyclosporine;

FIG. 3 f is a representation of the cyctotoxicity to A431 cell linesafter incubation with GCHQ488; and

FIG. 3 g is a representation of the cyctotoxicity to A431 cell linesafter incubation with GCHQ488 and prednisolone in a 1:5 ratio.

EXAMPLES Example 1

Materials

Glycol chitosan, palmitic acid N-hydroxysuccinimde, methyl iodide,1-bromohexadecane, pyrene and paclitaxel were all obtained from SigmaAldrich Co, UK. Prednisolone acetate, prednisolone and cyclosporine wereall obtained from Allergan, USA. Hydrochloric acid was obtained fromMerck, UK and all organic solvents were purchased from the Department ofPure and Applied Chemistry, University of Strathclyde.

Methods

Degradation of Glycol Chitosan (GC)

Acid degradation of glycol chitosan (GC) was carried out as previouslydescribed¹⁵. Glycol chitosan (2 g) was dissolved in hydrochloric acid (4M, 150 mL) and the solution filtered to remove insoluble impurities. Thefiltered solution was placed in a pre-heated water bath set at 50° C.After 48 h the reaction was stopped and the products isolated andpurified as described below. The reaction solution was exhaustivelydialysed (Visking Seamless Cellulose Tubing, molecular weight cutoff=12,400 for cytochrome c) against distilled water (5 L with 6 changesover 24 h). The dialysate at the end of the dialysis procedure had aneutral pH. The dialysate was subsequently freeze-dried and the materialwas recovered as cream coloured cotton wool like material. The aciddegradation step was repeated for either 4 h, 8 h, 24 h or 48 h to givematerials, which were further depolymerised.

Synthesis of Low Molecular Weight Palmitoyl Glycol Chitosan (PGC)

Palmitoyl glycol chitosan (PGC) was synthesised as previouslydescribed¹⁴. Glycol chitosan (500 mg), sodium bicarbonate (376 mg) in amixture of absolute ethanol (24 mL) and water (76 mL) was reacted withpalmitic acid N-hydroxysuccinimide (198 mg) in absolute ethanol (150mL). Palmitic acid N-hydroxysuccinimide solution was added dropwise. Theproduct was isolated after stirring for 72 h by evaporating off most ofthe ethanol, extraction of the remaining liquid with three volumes ofdiethyl ether (100 mL), exhaustive dialysis against water and freezedried to give a white cotton wool like solid.

Synthesis of Low Molecular Weight Hexadecyl Glycol Chitosan (GCH)

Hexadecyl glycol chitosan (GCH) was synthesised based on a modificationof the method of Wakita and Hashimoto²⁹. Glycol chitosan (500 mg) wasdissolved in a 1:1 mixture of methanol and N-methyl pyrrolidinone (20mL) and to this was added drop wise a solution of sodium bicarbonate(376 mg) dissolved in absolute ethanol (20 mL). 1-bromhexadecane (3.5mL) was freshly dissolved in a mixture of methanol, N-methylpyrrolidinone, absolute ethanol (5 mL:5 mL:10 mL) and this solution wasadded to the basic glycol chitosan solution drop wise over a 1 h timeperiod. The resulting reaction mixture was refluxed at 70-85° C. in anoil bath with stirring over a period of 4 hours and then filtered andthe filtrate retained. The ethanol was then evaporated off under reducedpressure at 50° C. and the residue produced dissolved in distilled water(30 mL). This solution was then dialysed against 5 L of water with 6changes over 24 hours and freeze dried to give an off white solid.

Synthesis of Low Molecular Weight Quaternary Ammonium Palmitoyl GlycolChitosan (GCPQ) and Low Molecular Weight Quaternary Ammonium HexadecylGlycol Chitosan (GCHQ)

Quaternisation was carried out using essentially the same method asreported by Domard and others³⁰. PGC or GCH (300 mg) was dispersed inN-methyl-2-pyrrolidone (25 ml) overnight for 12 h at room temperature.Sodium hydroxide (40 mg), methyl iodide (1.0 g) and sodium iodide (45mg) were added and the reaction stirred under a stream of nitrogen at36° C. for 3 h. The quaternary ammonium product was recovered byprecipitation with diethyl ether, filtered and washed with copiousamounts of absolute ethanol followed by copious amounts of diethyl etherto give a brown hygroscopic solid. The solid was dissolved in water (100ml) to give a yellow viscous solution. The resultant aqueous solutionwas exhaustively dialysed against water (5 L) with six changes over a 24h period and the product freeze-dried to give a white cotton-like solidwhich was present as the iodide salt. The quaternary ammonium iodide wasthen dissolved in water (150 ml) to give a clear solution and thesolution passed through a column (1×6 cm) packed with Amberlite IRA-93Cl⁻. Before use the column was packed with resin and subsequently withone volume of the resin (30 ml) and subsequently washed withhydrochloric acid solution (90 ml, 1 M) followed by distilled water (500ml) to give a neutral pH. The clear eluate from the column wasfreeze-dried to give GCPQ as a transparent fibrous solid.

¹H NMR

¹H NMR scans (with integration) and ¹H correlation spectroscopyexperiments were performed on GCPQ and GCHQ samples solubilised indeuterated methanol and the level of palmitoylation and quaternisationdetermined by integrating the palmitoyl methyl, alkyl methyl orquaternary ammonium methyl peaks relative to the sugar peaks³¹. The mole% palmitoylation, alkylation or quaternisation refers to the number ofmoles of sugar monomers bearing a palmitoyl, alkyl or quaternaryammonium group per 100 moles of sugar monomers respectively.

MW Determination

The molecular weight of degraded glycol chitosan (GC) was determined byGPC-MALLS (i.e. gel permeation chromatography—multi angle laser lightscattering). Polymers were dissolved in an acetate buffer (sodiumacetate 0.3M, acetic acid 0.2M) and filtered (0.2 μm) samples (200 μL,1-2 l mg mL ⁻¹) injected onto GPC columns using a Waters 717 plusAutosampler. Samples were chromatographed over a PSS HEMA-BIO 300 (330×8mm, particle size=10 μm, exclusion limit for dextran=5×10⁵) and a PSSHEMA-BIO 40 (330×8 mm, particle size=10 μm, exclusion limit fordextran=3×10⁶) column (Polymer Standards Services, Mainz, Germany). Themobile phase was an acetate buffer (sodium acetate 0.3M, acetic acid0.2M) and molecular weights were determined using a DAWN EOS MALLSdetector (18 angles at 20-150°—Wyatt Technology, USA) equipped with a 30mV linearly polarized gallium arsenide laser (λ=690 nm) and an OptilabDSP interferometric refractometer (λ=690 nm, Wyatt Technology, USA). Allthe measurements were carried out at room temperature. Molecularweights, molecular weight distribution and molecular size in solutionwere obtained from GPC graphs using Astra for Windows (v4.73) software.

The refractive index increment (dn/dc) of GC in the mobile phase wasmeasured with an Optilab DSP interferometric refractometer (λ=690 nm,Wyatt Technology, USA,) at 25° C. A Rheodyne 7725 sample injector wasused to load filtered (0.45 μm) polymer solutions of variousconcentrations and the data were processed using DNDC for Windows(v5.31) software.

Fluorescence Spectroscopy

A dilute aqueous solution of pyrene (2 μM) was prepared by initiallydissolving pyrene in ethanol (0.4 mg/mL). 100 μl of this solution waspipetted into a volumetric flask (100 mL) and the ethanol dried under astream of nitrogen gas. The solution was then made up in distilledwater. Using the aqueous pyrene solution as the solvent, polymersolutions were made at various concentrations. The fluorescence emissionspectra were recorded (340 nm-600 nm) at an excitation wavelength of 335nm. The I₃/I₁ ratio was calculated from the intensity of the third (383nm) and first (375 nm) vibronic peaks in the pyrene emission spectra³²and an increase in the size of the I₃ peak relative to the I₁ peakindicates a more hydrophobic environment³². In polar solvents such aswater this value is approximately 0.67.

Solubilisation Studies

Various levels of the GCPQ and GCHQ samples were dissolved in water anda weighed amount of drug added with probe sonication (MSE Soniprep 150,Sanyo, UK with the instrument set at 60-85% of its maximum output) forabout 5 minutes or until a clear solution is obtained. The presence of aclear solution was verified by optical density measurements (λ=600 nm,UV1 Spectrophotometer, ThermoUnicam, UK). The samples were stored atrefrigeration (4-8° C.) or room (22° C.) temperature. At various timeintervals liquid samples were filtered (0.45 μm, 25 mm in diameter) andthe first millilitre discarded. The subsequent filtrate was retained andanalysed for dissolved drug using HPLC.

Assay for Prednisolone

Prednisolone levels were analysed in filtered samples of thepolymer—drug formulation by HPLC. Samples (20 μL), appropriately dilutedwith the mobile phase (acetonitrile, water 36:64) were injected onto areverse phase Symmetry C18, 3.5 μm column (4.6 mm×755 mm, WatersInstruments, UK) by means of a Waters 717 autosampler and a Waters 515isocratic pump. Peak detection was via a Waters 486 variable wavelengthUV detector with the wavelength set at 243 nm and data was collectedusing a Waters 746 data module. The mobile phase was set at a flow rateof 1 ml min⁻¹. A standard curve was prepared with samples ofprednisolone solubilised in the mobile phase (0.1-1.0 mg Ml⁻¹).

Assay for Cyclosporine

Cyclosporine was analysed by HPLC on the same instrumentation as aboveexcept that the column was a Waters Spherisorb 5 μm, 4.6 mm×250 mmcolumn, maintained at 80° C. with a Jones Chromatography Column Heatermodel 7971. Filtered Samples (20 μL) dissolved in acetonitrile, water(1:1) were injected onto the column and the mobile phase wasacetonitrile:water:tert-butyl-methyl-ether:phosphoric acid(600:350:50:1) at a flow rate of 1.2 mL min⁻¹. Peaks were detected by UVdetection at a wavelength of 210 nm. A standard curve was prepared usingsolutions of the drug (1-10 μg mL⁻¹).

Haemocompatibilty Studies

Approximately 5 ml of human blood was centrifuged (1000 g×10 min), thesupernatant removed and the erythrocyte pellet recovered. The pellet waswashed twice by resuspending in PBS (pH 7.4, 4° C.) and centrifuging(1000 g×10 min). The pellet was then weighed and a 3% w/w dispersion ofthe erythrocytes was prepared in PBS (pH 7.4). 100 μL of thiserythrocyte suspension was placed into each well of a 96 well plate. Tothis erythrocyte suspension was added 100 μL of varying concentrationsof the sample formulations. Sample formulations were either prepared inphosphate buffered saline (PBS, pH=7.4) or water. PBS (pH=7.4) andTriton X-100 (1% w/v) served as negative and positive controlsrespectively. The plate was incubated at 37° C. for 4 h after which theplate was centrifuged (1000 g×10 min) and 100 μL of the supernatantremoved and placed in a new microtitre plate. The absorbance wasmeasured at 570 nm and the results expressed as percentage haemolysisassuming Triton X-100 gave 100% haemolysis and PBS (pH=7.4) gave 0%haemolysis.

Cytotoxicity Studies

A human lung carcinoma cell line (A549, ATCC CCL-185) and a humanepidermoid carcinoma cell line (A431, ATCC CRL-1555) were bothmaintained in Dulbecco's minimum essential medium (DMEM) supplementedwith 10% foetal calf serum (FCS) and 2 mM glutamine (GibcoBRL, U.K.) at10% CO₂ and 37° C.

To measure the cytotoxicity of the formulations, a standard MTT(3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide thiazolylblue indicator dye) assay was carried out³³. 96 well microtitre plates,1 per formulation, were seeded with about 800 cells per well andincubated at 37° C. with 10% CO₂ for 72 hours. The medium was thenremoved from the wells and varying concentrations of each formulation(200 μL, prepared in 6% dextrose and diluted in medium) were added tothe wells. DMEM alone and Triton X-100 (1% w/v) were used as positiveand negative controls respectively. The plates were incubated under thestandard conditions for 4 hours and the drug solutions then removed fromthe wells, replaced with DMEM and the cells further incubated for 72hours. The indicator dye (50 μL, 50 mg mL⁻¹) was then added to thecells, which were subsequently incubated in the dark for a further 4hours. At the end of the incubation time, the dye was removed and thecells lysed by addition of dimethylsulfoxide (200 μL). To the lysedcells was then added Sorensen's glycine buffer (25 μL) and theabsorbance measured at 570 nm. Values are expressed as a percentage ofthe control minus the background obtained from the wells containing justDMEM.

The structures of GCPQ and GCHQ are shown in FIGS. 1 a and 1 b,respectively. GCPQ and GCHQ were recovered as white fibrous solids. Withthe glycol chitosan starting material as has been reported previously¹⁵,an increase in the acid degradation time led to an increase in thedegree of depolymerisation (Table 1), however after the initial 48 hthere is a slowing down in the level of depolymerisation (Table 1).

TABLE 1 Molecular Weights of Glycol Chitosan Samples Acid DegradationMolecular Polydispersity POLYMER Time Weight (kD) Mw/Mn GC0  0 h 1041.05 GC48 48 h 19.3 1.20 GC484 48 h + 4 h  17.9 1.23 GC488 48 h + 8 h 17.2 1.28 GC4824 48 h + 24 h — — GC4848 48 h + 48 h 15.2 1.08

The lipidic and quaternary ammonium derivatives of these glycol chitosanpolymers are able to solubilise hydrophobic drugs such as prednisoloneand cyclosporine in aqueous media as shown in Table 2.

TABLE 2 Solubilisation by Glycol Chiosan Polysoaps* Number of Number ofQuaternary Hyrophilicity Molecular Palmitoyl or Ammonium Index (HI) =Weight (kD) Hexadecyl* Groups per 100 Mole % quaternisation/ MaxixmumMaximum of Glycol Groups per 100 sugar Mole % palmitoylation Level ofLevel of GC Acid Chitosan sugar monomers Monomers OR Mole % PrednisoloneCyclosporin Sample Degradation starting (mole % (mole % Quaternisation/Solubilised Solubilised Name Time (h) material hydrophobisation)Quaternisation) Mole % Cetylation (mg mL⁻¹) (mg mL⁻¹) GCPQ484 4 17.9 7.711.3 1.47 Polymer did not polymer did not dissolve dissolve GCPQ488 817.2 3.0 10.0 3.33 0.8 1.0 GCPQ4824 24 — 3.6 16.0 4.44 1.0 1.0 GCPQ484848 15.2 8.2 13.0 1.59 1.0 0.5 GCHQ484 4 17.9 3.0* 7.9 2.63 Polymer didnot Polymer did not dissolve dissolve GCHQ488 8 17.2 4.5* 19.0 4.22 5 1CCHQ4824 24 — — — — 0.5 0.5 *All polymers were present at a level of 1mg mL⁻¹

All solutions were clear transparent liquids with optical densityreadings of less than 0.001 against distilled water which had an opticaldensity of 0.000.

Solutions remained optically clear on dilution ten times with water.These solutions are in contrast to the chitosan—paclitaxel colloidsreported by Miwa and others¹¹ where liquids with “turbidity” wereobtained and the hydrophobic drug was added to the polymer formulationin the presence of 10% v/v of ethanol, with the removal of ethanol beingattempted but not confirmed.

¹H NMR was used to quantify the level of quaternisation, acylation andalkylation and a value for the hydrophilicity index (HI) is quoted inTable 2. The HI (derived from the ¹H NMR data) is a term used here tocharacterise the hydrophilicity of the polymers and this valuequantifies the relationship between additional ionic hydrophilicsubstituents and hydrophobic substituents added to a previously aqueoussoluble polymer or aqueous soluble polymer derivative. Aqueous solublepolymers are described as polymers soluble in water at a level of above1 mg mL⁻¹⁸. These carbohydrate polymers may be additionally substitutedwith non-ionic hydrophilic units as is shown here.

The data in Table 3 shown below, shows that the polymers aggregate inaqueous media to produce hydrophobic domains as evidenced by theincrease in the I₃/I₁ ratio.

TABLE 3 I3/I1 data for GCPQ and GCHQ Polymers Polymer I3/I1Concentration GCPQ488B GCPQ4824B GCPQ4848B GCHQ488B GCHQ4824B 0 0.670.67 0.67 0.67 0.67 0.25 — — 0.91 — — 0.5 0.67 0.82 0.88 0.80 0.85 10.97 0.85 1.02 0.86 1.02 1.5 1.02 — — 0.91 1.18 2.0 1.28 0.86 0.92 11.25

[B merely means a different batch of polymer]

These hydrophobic domains provide areas for the solubilisation ofhydrophobic solutes while the hydrophilic substituents increase theaffinity of the polymer with the aqueous solvent and prevent phaseseparation.

As shown in Table 3 the hydrophobicity of the polymer aggregates whenpresent at a polymer concentration of 1 mg mL⁻¹ (as determined using apyrene probe) followed the trendGCHQ4824=GCPQ4848>GCPQ488>GCHQ488=GCPQ4824. This hydrophobicity rankingobtained by the use of the pyrene probe is similar to the hydrophobicityranking obtained using the HI: GCPQ4848=1.58, GCPQ488=3.33, GCHQ488#1(i.e. different batch of polymer)=4.22 and GCPQ4824=4.44. (Table 2).Additionally the maximum level of drug solubilised in the case of themore polar drug prednisolone is solubilised by GCHQ488 (Tables 2&4), oneof the more polar polysoaps (Table 2).

TABLE 4 Prednisolone Stability Studies Original Polymer PrednisolonePrednisolone Concentration Age of Storage Concentrations DetectedPolymer (mg mL⁻¹) Formulation Conditions (mg mL⁻¹) (mg mL⁻¹) GCPQ488 115 weeks RT 1.0 0.2 GCPQ488 1 15 weeks 4° C. 0.75 0.2 GCHQ488 1 12 weeksRT 5.0 4.5 GCHQ488A* 1 2 weeks 4° C. 2.5 2.4 GCHQ488A* 1 10 weeks 4° C.2.5 2.4 GCHQ488A* 1 2 weeks 4° C. 5.0 4.4 GCHQ488A* 1 10 weeks 4° C. 5.04.3 *= different batch of GCHQ488

Also the least polar polysoaps (with the lowest HI value) and thehighest molecular weight i.e.—GCPQ484 and GCHQ484 are insoluble in waterat a level of 1 mg mL⁻¹ (Tables 1 and 2).

Relatively high levels of hydrophobicity also hamper (albeit to a lesserextent) the solubilisation of cyclosporine, as evidenced by the ratherlow maximum levels of cyclosporine solubilised by GCPQ4848 and GCHQ4824.This is shown in Table 5 below.

TABLE 5 Cyclosporine Stability Studies Initial Level Cyclo- Polymer ofsporine Concen- Age of Storage Cyclosporine Detected Lymer trationsformulation Conditions (mg mL⁻¹) (mg mL⁻¹) GCPQ488 1 1 day 4° C. 1 0.95GCPQ488 1 3 days 4° C. 1 0.92 GCPQ488 1 1 week 4° C. 1 1.10 GCPQ488 1 2weeks 4° C. 1 1.10 GCPQ488 1 4 weeks 4° C. 1 0.90 GCPQ488 1 8 weeks 4°C. 1 0.80 GCPQ488 1 12 weeks 4° C. 1 0.70 GCPQ488 1 15 weeks 4° C. 10.80 GCPQ488 1 22 weeks 4° C. 1 0.70

The solubilising polymer must thus be depolymerised as well assubstituted with a controlled number of hydrophobic and hydrophilicmoieties. Within the molecular weight range (10-20 kD), HI (whencalculated from the ¹H NMR) values of above about 2 appear to be themost efficient polymers in the case of the more polar palmitoylsubstituent and HI values above about 3 appear to be the most efficientsolubilisers in the case of the less polar hexadecyl substituents (Table2). Clearly in the case of prednisolone and cyclosporine, a high levelof quaternisation and low level of hydrophobic modification favourssolubilisation with these polymers (Tables 1-4).

With the N-lauroyl 6-carboxymethyl chitosan polymers preparedpreviously¹¹, a shift to a higher level of the carboxymethyl groups anda lower level of lauroyl groups resulted in a decreased association ofpaclitaxel with the chitosan based colloid. In the present invention,the ability of the polymer molecule to be fully hydrated by watermolecules (presence of ionic quaternary ammonium groups) plays a moreimportant role than the ability of the polymer to aggregate intopolymeric micelles (presence of hydrophobic groups) and provide ahydrophobic cavity within which hydrophobic solutes may be shielded.

The solubilising polymer should be prepared from an aqueous soluble anddepolymerised carbohydrate and the most effective polymers have a degreeof polymerisation of less than 200 monomer units. The solubilisingpolymer may further have a level of hydrophobic substitution notexceeding 10 substituents per every 100 monomers and finally anadditional ionic hydrophilic substitution level of equal to or more than1 substituent per polymer chain. There may be an optional additionalhydrophilic substituent of at least 1 hydrophilic substituent perpolymer chain. Previous work with N-lauroyl 6-carboxymethyl chitosan hasshown that there is no increase in the association of paclitaxel withthe chitosan based colloid on reducing the degree of polymerisation froma molecular weight of 50 kD to 2 kD¹¹. However, it is shown herewiththat even small changes in molecular weight affect the solubilisingproperties of the polymers as evidenced by the data presented on GCPQ484and GCPQ4848 (Table 2).

Finally, in the work described using N-lauroyl 6-carboxymethylchitosan,polymers encapsulating paclitaxel had a level of hydrophobicsubstitution exceeding 20 substituents per 100 monomers and a minimumlevel of 140 carboxymethyl groups per 100 monomers¹¹. In contrast thepresent invention relates to a maximum level of 10 hydrophobicsubstituents per 100 monomer units.

The solubilising polymer of the present invention may contain additionaltargeting groups such as peptides, antibodies and other ligands e.g.folate and transferrin ligands which will allow the polymer to targetendogenous receptors and thus target its drug payload to such endogenousreceptors at the site of pathology.

With the HPLC assays used the retention time of prednisolone was 2.7 minand the standard curve had a correlation coefficient of 0.998 while theretention time for cyclosporine was 13.7 min and the standard curve hada correlation coefficient of 0.98. Solubilised material was stable forup to 12 weeks with chitosan based polymer formulations retaining up to9.0% of solubilised drug for this length of time (Tables 4 and 5).

The solubilising polymers presented herein are biocompatible. Solutionsof the GCPQ488 and GCPQ488, cyclosporine samples in water resulted in amaximum 40% cell lysis, while formulations within isotonic PBS (pH=7.4)gave about 10% cell lysis up to a polymer concentration of 1 mg mL⁻¹(FIG. 2). The hypotonic water environment is thus responsible for theobserved cell lysis. While the inclusion of up to 1 mg mL⁻¹ cyclosporinewithin polymer formulations appeared to protect the cells from lysis(especially in the presence of water as the solvent), the inclusion of 5mg mL⁻³ prednisolone increased the level of lysis observed with thepolymers (FIG. 2). It is concluded that these glycol chitosan polysoapsshow no appreciable levels of haemolysis to cells. However, what shouldbe noted here is the observation of drug activity in the presence of thepolysoaps.

Below a level of 0.1 mg mL⁻¹ none of the glycol chitosan based polysoapsare particularly cytotoxic in both the A431 and the A549 cell lines(FIG. 3). However the addition of either prednisolone or cyclosporinedoes cause the appearance of some cytotoxicity although none of theformulations resulted in the observation of less than 50% cell survivalbelow a polymer concentration of 0.01 mg mL⁻¹. It may be concluded thatthe glycol chitosan polysoaps are not particularly cytotoxic polymersand any toxicity seen with the drug formulations is largely due to theaddition of drug and not due to the toxicity of the polymer per se. Onceagain what should be noted here is the observation of drug activity inthe presence of the polysoaps.

Example 2

This Example relates to a high throughput method of selection ofcarbohydrate solubilisers

Materials

Glycol chitosan, palmitic acid N-hydroxysuccinimide, methyl iodide,N-methyl pyrrolidone, sodium iodide, sodium hydroxide, prednisolone and6-methyl prednisolone were all obtained from Sigma-Aldrich Co. UK.Absolute ethanol was supplied by Bamford Laboratories, UK and diethylether by BDH Laboratories, UK. Acetonitrile was supplied by Riedelde-Haen, Germany.

Methods

Degradation of Glycol Chitosan (GC)

Glycol chitosan (GC) was degraded for 48 h as described above¹⁵. 25quaternary ammonium palmitoyl glycol chitosan polymers were synthesisedfrom the degraded material with differing levels of hydrophobic(palmitoyl) and hydrophilic (quaternary ammonium) substitution.

Solutions of palmitic acid N-hydroxysuccinimide (PNS) in ethanol (5.28mg mL⁻¹), sodium iodide (2 mg mL⁻¹) in N-methyl pyrrolidone (NMP),sodium hydroxide in absolute ethanol (10 mg mL⁻¹), glycol chitosan (10mg mL⁻¹, GC) in a solution of sodium bicarbonate (7.53 mg mL⁻¹) andprednisolone (10 mg mL⁻¹) in methanol were prepared.

25 different polymers were synthesised by adding a solution of palmiticacid N-hydroxysuccinimide at the levels shown in Table 1 to the glycolchitosan—sodium bicarbonate solution (1 mL) contained in a 25 mL testtube. The tubes were shaken for 16 h at room temperature andsubsequently heated at 85° C. for 4 h to evaporate off the ethanol.

The residues in the tubes were extracted with diethyl ether (3×15 mL) toremove unreacted palmitic acid and subsequently washed with absoluteethanol to remove polar contaminants. Ethanol was removed and residualethanol was dried under a stream of nitrogen. For the quaternisationreaction the solution of sodium iodide in NMP was added to the testtubes in the quantities shown in Table 1. This was followed by theaddition of the solution of sodium hydroxide and the addition of methyliodide in the quantities shown in Table 1.

The tubes were heated for 3 h at 36° C. and diethyl ether added to thetubes (5 mL). This caused the quaternary ammonium product toprecipitate. The supernatant was decanted from the tubes and theresidues washed with diethyl ether (3×5mL). The residue was then left todry overnight and water (2 mL) subsequently added to each tube toproduce polymer solutions known herein as “concentrated polymersolutions”. To a separate set of 25 tubes was added 0.1 mL of theprednisolone solution in methanol. Methanol was removed under a streamof nitrogen and to each tube was added a sample of one of the 25quaternary ammonium palmitoyl glycol chitosan solutions (1 mL) fromabove.

This mixture was probe sonicated (MSE Instruments, Sanyo, UK) filteredusing a 0.45 μm filter and analysed by high performance liquidchromatography (HPLC). The polymer solutions obtained from the synthesisstep above were diluted by adding to 1 mL each of the 25 solutions to anadditional volume of water (1 mL) to produce what are termed here as“dilute polymer solutions” and the prednisolone solubilisation proceduredescribed above repeated once more with a more dilute solution of thesynthesised polymer.

Prednisolone levels were analysed in filtered (0.45 μm) samples of thepolymer—drug formulation by HPLC. Samples (20 μL), appropriately dilutedwith the mobile phase (acetonitrile, water 36:64) and containing6-methyl prednisolone (1 μg mL⁻¹) were injected onto a reverse phaseSymmetry C18, 3.5 μm column (4.6 mm×75 mm, Waters Instruments, UK) bymeans of a Waters 717 autosampler and a Waters 515 isocratic pump. Peakdetection was via a Waters 486 variable wavelength UV detector with thewavelength set at 243 nm and data was collected using Waters Empowersoftware. The mobile phase was set at a flow rate of 1 ml min⁻¹. Astandard curve was prepared with 6-methyl prednisolone (1 μg mL−1) asthe internal standard and with samples of prednisolone solubilised inthe mobile phase (0.1-20 μg mL⁻¹)

TABLE 6 High throughput polymer synthesis–reagent volumes (mL)Increasing methyl iodide Increasing palmitic acid N- (hydrophilichydroxysuccinimide (hydrophobic substitution) substitution) PolymerPolymer Polymer Polymer Polymer E1 E2 E3 E4 E5 PNS - 0.5 PNS - 1 PNS - 2PNS - 3 PNS - 4 NaI - 3.0 NaI - 3.0 NaI - 3.0 NaI - 3.0 NaI - 3.0 NaOH -0.6 NaOH - 0.6 NaOH - 0.6 NaOH - 0.6 NaOH - 0.6 CH₃I - 0.062 CH₃I -0.062 CH₃I - 0.062 CH₃I - 0.062 CH₃I - 0.062 Polymer Polymer PolymerPolymer Polymer D1 D2 D3 D4 D5 PNS - 0.5 PNS - 1 PNS - 2 PNS - 3 PNS - 4NaI - 2.5 NaI - 2.5 NaI - 2.5 NaI - 2.5 NaI - 2.5 NaOH - 0.5 NaOH - 0.5NaOH - 0.5 NaOH - 0.5 NaOH - 0.5 CH₃I - 0.052 CH₃I - 0.052 CH₃I - 0.052CH₃I - 0.052 CH₃I - 0.052 Polymer Polymer Polymer Polymer Polymer C1 C2C3 C4 C5 PNS - 0.5 PNS - 1 PNS - 2 PNS - 3 PNS - 4 NaI - 2 NaI - 2 NaI -2 NaI - 2 NaI - 2 NaOH - 0.4 NaOH - 0.4 NaOH - 0.4 NaOH - 0.4 NaOH - 0.4CH₃I - 0.042 CH₃I - 0.042 CH₃I - 0.042 CH₃I - 0.042 CH₃I - 0.042 PolymerPolymer Polymer Polymer Polymer B1 B2 B3 B4 B5 PNS - 0.5 PNS - 1 PNS - 2PNS - 3 PNS - 4 NaI - 1.5 NaI - 1.5 NaI - 1.5 NaI - 1.5 NaI - 1.5 NaOH -0.3 NaOH - 0.3 NaOH - 0.3 NaOH - 0.3 NaOH - 0.3 CH₃I - 0.032 CH₃I -0.032 CH₃I - 0.032 CH₃I - 0.032 CH₃I - 0.032 Polymer Polymer PolymerPolymer Polymer A1 A2 A3 A4 A5 PNS - 0.5 PNS - 1 PNS - 2 PNS - 3 PNS - 4NaI - 1 NaI - 1 NaI - 1 NaI - 1 NaI - 1 NaOH - 0.2 NaOH - 0.2 NaOH - 0.2NaOH - 0.2 NaOH - 0.2 CH₃I - 0.022 CH₃I - 0.022 CH₃I - 0.022 CH₃I -0.022 CH₃I - 0.022 All values are in ml.Results

TABLE 7 High throughput polymer synthesis–prednisolone solubilities withconcentrated polymer solutions (mg mL⁻¹) Increasing methyl iodideIncreasing palmitic acid N- (hydrophilic hydroxysuccinimide (hydrophobicsubstitution) substitution) Polymer Polymer Polymer Polymer Polymer E1E2 E3 E4 E5 0.472 0.613 0.632 — — Polymer Polymer Polymer PolymerPolymer D1 D2 D3 D4 D5 0.984 0.978 0.828 0.847 0.906 Polymer PolymerPolymer Polymer Polymer C1 C2 C3 C4 C5 0.433 0.374 0.346 0.399 0.374Polymer Polymer Polymer Polymer Polymer B1 B2 B3 B4 B5 0.373 0.166 0.1280.369 0.367 Polymer Polymer Polymer Polymer Polymer A1 A2 A3 A4 A5 0.5980.590 — — 0.512

TABLE 8 High throughput polymer synthesis–prednisolone solubilities withdilute polymer solutions (mg mL⁻¹) Increasing methyl iodide Increasingpalmitic acid N- (hydrophilic hydroxysuccinimide (hydrophobicsubstitution) substitution) Polymer Polymer Polymer Polymer Polymer E1E2 E3 E4 E5 0.987 0.830 — — — Polymer Polymer Polymer Polymer Polymer D1D2 D3 D4 D5 0.954 0.802 0.847 0.713 0.633 Polymer Polymer PolymerPolymer Polymer C1 C2 C3 C4 C5 0.831 0.700 0.625 0.776 0.731 PolymerPolymer Polymer Polymer Polymer B1 B2 B3 B4 B5 0.816 0.785 0.860 0.6800.626 Polymer Polymer Polymer Polymer Polymer A1 A2 A3 A4 A5 0.993 0.9560.691 0.644 0.646Comment on Results

The high through put method outlined above was able to select polymerswith a high solubilising ability (e.g. Polymer E1) for a particular drugwhich in this case was prednisolone. Polymer E1 may then be synthesisedin bulk quantities. Lower concentrations of the polymer (dilute polymersolutions) of about 0.01-5 m m⁻¹ were superior solubilisers of the modeldrug than higher concentrations (of about 5-10 mg mL⁻¹) of the polymer.

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1. A solubilising carbohydrate polymer of average molecular weight ofabout 2-30 kD according to the following formula:

wherein m is 0.01% to 10.00%; n is 0.01% to 99.98%; p is 0.00% to 99.98%X is any hydrophobic, linear, branched or cyclo form of an alkyl,alkenyl, alkynyl, aryl, amine, amide, alcohol or acyl group; R′, R″, R′″are independently any linear, branched or cyclo form of an alkyl,alkenyl, alkynyl, aryl, acyl group, a sugar substituent selected fromglucose, galactose, fructose and muramic acid, or oligo polyoxa C₁-C₃alkylene units, optionally substituted with amine, amide or alcohol; andR₁, R₂ and R₃ are independently any linear, branched, or cyclo forms ofany alkyl, alkenyl, alkynyl, aryl or acyl group.
 2. A solubilisingcarbohydrate polymer according to claim 1 wherein the m, n and p unitsform any arrangement in the solubilising carbohydrate polymer.
 3. Asolubilising carbohydrate polymer according to claim 1 wherein thearrangement of the m, n and p units are random or in a block copolymerform.
 4. A solubilising carbohydrate polymer according to claim 3wherein the block copolymer is mnpmnpmnp.
 5. A method of forming asolubilising carbohydrate polymer according to claim 1 wherein themethod comprises; depolymerising a carbohydrate polymer to formdepolymerised carbohydrate; reacting the depolymerised carbohydrate witha first reactive compound to form hydrophobic side-groups on thecarbohydrate backbone and thus form hydrophobically substituteddepolymerised carbohydrate; and adding a second reactive compound to thehydrophobically substituted depolymerised carbohydrate to quaternarisean amine group and thereby form the solubilising carbohydrate polymer.6. A method according to claim 5 wherein the carbohydrate polymer isselected from the following: glycol chitosans, dextrans, alginic acids,starches, dextran and guar gums.
 7. A carbohydrate polymer according toclaim 1 and a pharmaceutically acceptable carrier.
 8. A carbohydratepolymer according to claim 7 wherein the ratio of carbohydrate polymerto pharmaceutical acceptable carrier ranges from 0.05 wt. % to 10 wt. %.9. A pharmaceutical composition comprising a carbohydrate polymeraccording to claim 1 and a drug.
 10. A pharmaceutical compositionaccording to claim 9 wherein the drug is selected from any of thefollowing: prednisolone; cyclosporine; oestradiol, testosterone, drugswith multicyclic ring structures which lack polar groups etoposide. 11.A method of dissolving poorly soluble drugs in a carbohydrate polymerwherein the solubilising carbohydrate polymer is as defined in claim 1,whereby on dissolving a poorly soluble drug in the solubilisingcarbohydrate polymer a substantially clear solution is obtained.
 12. Amethod according to claim 11 wherein the solubilising carbohydratepolymer is selected from any derivatives of the following: chitosans,dextrans, alginic acids, starches, dextran and guar gums.
 13. Apharmaceutical composition according to claim 10 wherein the drug ispaclitaxel.
 14. A solubilising carbohydrate polymer according to claim1, wherein X is selected from any of the following linear or branched orcyclo groups: C₁-C₃₀; C₈-C₂₄; or C₁₂-C₁₈.
 15. A solubilisingcarbohydrate polymer according to claim 1, wherein X is CH₃(CH₂)₁₄CONH,or CH₃(CH₂)₁₅NH.
 16. A solubilising carbohydrate polymer according toclaim 1, wherein R′, R″ and R″′ are independently any linear or branchedor cyclo form of the following alkyl, alkenyl, alkynyl, aryl, or acylgroups: C₁-C₃₀; C₁-C₁₂; and C₁-C₆, optionally substituted with amine,amide or alcohol.
 17. A solubilising carbohydrate polymer according toclaim 1, wherein R′, R″ and R′″ are C₁-C₄ linear glycol based groups.18. A solubilising carbohydrate polymer according to claim 1, whereinR′, R″ and R′″ are any of the following sugar substituents: glucose,galactose, fructose and muramic acid.
 19. A solubilising carbohydratepolymer according to claim 1, wherein R′, R″ and R′″ are oligo polyoxaC₁-C₃ alkylene units.
 20. A solubilising carbohydrate polymer accordingto claim 1, wherein all of R′, R″ and R′″ are CH₂OCH₂CH₂OH or CH₂CH₂OH.